Microfluidic Device For Cell Separation And Uses Thereof

ABSTRACT

The invention features methods for separating cells from a sample (e.g., separating fetal red blood cells from maternal blood). The method begins with the introduction of a sample including cells into one or more microfluidic channels. In one embodiment, the device includes at least two processing steps. For example, a mixture of cells is introduced into a microfluidic channel that selectively allows the passage of a desired type of cell, and the population of cells enriched in the desired type is then introduced into a second microfluidic channel that allows the passage of the desired cell to produce a population of cells further enriched in the desired type. The selection of cells is based on a property of the cells in the mixture, for example, size, shape, deformability, surface characteristics (e.g., cell surface receptors or antigens and membrane permeability), or intracellular properties (e.g., expression of a particular enzyme).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/529,453, having a §371 date of Dec. 19, 2005, which is the NationalStage of PCT/US03/30965, filed Sep. 29, 2003, which claims benefit ofU.S. Provisional Application Nos. 60/414,065, 60/414,258, and60/414,102, filed on Sep. 27, 2002, each of which is hereby incorporatedby reference.

BACKGROUND OF THE INVENTION

The invention relates to the fields of medical diagnostics andmicrofluidics.

There are several approaches devised to separate a population ofhomogeneous cells from blood. These cell separation techniques may begrouped into two broad categories: (1) invasive methods based on theselection of cells fixed and stained using various cell-specificmarkers; and (2) noninvasive methods for the isolation of living cellsusing a biophysical parameter specific to a population of cells ofinterest.

Invasive techniques include fluorescence activated cell sorting (FACS),magnetic activated cell sorting (MACS), and immunomagnetic colloidsorting. FACS is usually a positive selection technique that uses afluorescently labeled marker to bind to cells expressing a specific cellsurface marker. FACS can also be used to permeabilize and stain cellsfor intracellular markers that can constitute the basis for sorting. Itis fast, typically running at a rate of 1,000 to 1,500 Hz, and wellestablished in laboratory medicine. High false positive rates areassociated with FACS because of the low number of photons obtainedduring extremely short dwell times at high speeds. Complicatedmultiparameter classification approaches can be used to enhance thespecificity of FACS, but multianalyte-based FACS may be impractical forroutine clinical testing because of the high cost associated with it.The clinical application of FACS is further limited because it requiresconsiderable operator expertise, is laborious, results in cell loss dueto multiple manipulations, and the cost of the equipment is prohibitive.

MACS is used as a cell separation technique in which cells that expressa specific surface marker are isolated from a mixture of cells usingmagnetic beads coated with an antibody against the surface marker. MACShas the advantage of being cheaper, easier, and faster to perform ascompared with FACS. It suffers from cell loss due to multiplemanipulations and handling. Moreover, magnetic beads often autofluoresceand are not easily separated from cells. As a result, many of theimmunofluorescence techniques used to probe into cellular function andstructure are not compatible with this approach.

A magnetic colloid system has been used in the isolation of cells fromblood. This colloid system uses ferromagnetic nanoparticles that arecoated with goat anti-mouse IgG that can be easily attached to cellsurface antigen-specific monoclonal antibodies. Cells that are labeledwith ferromagnetic nanoparticles align in a magnetic field alongferromagnetic Ni lines deposited by lithographic techniques on anoptically transparent surface. This approach also requires multiple cellhandling steps including mixing of cells with magnetic beads andseparation on the surfaces. It is also not possible to sort out theindividual cells from the sample for further analysis.

Noninvasive techniques include charge flow separation, which employs ahorizontal crossflow fluid gradient opposing an electric field in orderto separate cells based on their characteristic surface chargedensities. Although this approach can separate cells purely onbiophysical differences, it is not specific enough. There have beenattempts to modify the device characteristics (e.g., separator screens,buffer counterflow conditions, etc.) to address this major shortcomingof the technique. None of these modifications of device characteristicshas provided a practical solution given the expected individualvariability in different samples.

Since the prior art methods suffer from high cost, low yield, and lackof specificity, there is a need for a method for depleting a particulartype of cell from a mixture that overcomes these limitations.

SUMMARY OF THE INVENTION

The invention features methods for separating cells from a sample (e.g.,separating fetal red blood cells from maternal blood). The method beginswith the introduction of a sample including cells into one or moremicrofluidic channels. In one embodiment, the device includes at leasttwo processing steps. For example, a mixture of cells is introduced intoa microfluidic channel that selectively allows the passage of a desiredtype of cell, and the population of cells enriched in the desired typeis then introduced into a second microfluidic channel that allows thepassage of the desired cell to produce a population of cells furtherenriched in the desired type. The selection of cells is based on aproperty of the cells in the mixture, for example, size, shape,deformability, surface characteristics (e.g., cell surface receptors orantigens and membrane permeability), or intracellular properties (e.g.,expression of a particular enzyme).

In practice, the method may then proceed through a variety of processingsteps employing various devices. In one step, the sample is combinedwith a solution in the microfluidic channels that preferentially lysesone type of cell compared to another type. In another step, cells arecontacted with a device containing obstacles in a microfluidic channel.The obstacles preferentially bind one type of cell compared to anothertype. Alternatively, cells are arrayed individually for identificationof the cells of interest. Cells may also be subjected to size,deformability, or shape based separations. Methods of the invention mayemploy only one of the above steps or any combination of the steps, inany order, to separate cells. The methods of the invention desirablyrecover at least 75%, 80%, 90%, 95%, 98%, or 99% of the desired cells inthe sample.

The invention further features a microfluidic system for the separationof a desired cell from a sample. This system may include devices forcarrying out one or any combination of the steps of the above-describedmethods. One of these devices is a lysis device that includes at leasttwo input channels; a reaction chamber (e.g., a serpentine channel); andan outlet channel. The device may additional include another input and adilution chamber (e.g., a serpentine channel). The lysis device isarranged such that at least two input channels are connected to theoutlet through the reaction chamber. When a dilution chamber is present,it is disposed between the reaction chamber and the outlet, and anotherinlet is disposed between the reaction and dilution chambers. The systemmay also include a cell depletion device that contains obstacles thatpreferentially bind one type of cell when compared to another type,e.g., they are coated with anti-CD45, anti-CD36, anti-GPA, or anti-CD71antibodies. The system may also include an arraying device that containsa two-dimensional array of locations for the containment of individualcells. The arraying device may also contain actuators for the selectivemanipulation (e.g., release) of individual cells in the array. Finally,the system may include a device for size based separation of cells. Thisdevice includes sieves that only allow passage of cells below a desiredsize. The sieves are located with a microfluidic channel through which asuspension of cells passes, as described herein. When used incombination, the devices in the system may be in liquid communicationwith one another. Alternatively, samples that pass through a device maybe collected and transferred to another device.

By “a depleted cell population” is meant a population of cells that hasbeen processed to decrease the relative population of a specified celltype in a mixture of cells. Subsequently collecting those cells depletedfrom the mixture also leads to a sample enriched in the cells depleted.

By an “enriched cell population” is meant a population of cells that hasbeen processed to increase the relative population of a specified celltype in a mixture of cells.

By “lysis buffer” is meant a buffer that, when contacted with apopulation of cells, will cause at least one type of cell to lyse.

By “to cause lysis” is meant to lyse at least 90% of cells of aparticular type.

By “not lysed” is meant less than 10% of cells of a particular type arelysed. Desirably, less that 5%, 2%, or 1% of these cells are lysed.

By “type” of cell is meant a population of cells having a commonproperty, e.g., the presence of a particular surface antigen. A singlecell may belong to several different types of cells.

By “serpentine channel” is meant a channel that has a total length thatis greater than the linear distance between the end points of thechannel. A serpentine channel may be oriented entirely vertically orhorizontally. Alternatively, a serpentine channel may be “3D,” e.g.,portions of the channel are oriented vertically and portions areoriented horizontally.

By “microfluidic” is meant having one or more dimensions of less than 1mm.

By “binding moiety” is meant a chemical species to which a cell binds. Abinding moiety may be a compound coupled to a surface or the materialmaking up the surface. Exemplary binding moieties include antibodies,oligo- or polypeptides, nucleic acids, other proteins, syntheticpolymers, and carbohydrates.

By “obstacle” is meant an impediment to flow in a channel, e.g., aprotrusion from one surface.

By “specifically binding” a type of cell is meant binding cells of thattype by a specified mechanism, e.g., antibody-antigen interaction. Thestrength of the bond is generally enough to prevent detachment by theflow of fluid present when cells are bound, although individual cellsmay occasionally detach under normal operating conditions.

By “rows of obstacles” is meant is meant a series of obstacles arrangedsuch that the centers of the obstacles are arranged substantiallylinearly. The distance between rows is the distance between the lines oftwo adjacent rows on which the centers are located.

By “columns of obstacles” is meant a series of obstacles arrangedperpendicular to a row such that the centers of the obstacles arearranged substantially linearly. The distance between columns is thedistance between the lines of two adjacent columns on which the centersare located.

The methods of the invention are able to separate specific populationsof cells from a complex mixture without fixing and/or staining. As aresult of obtaining living homogeneous population of cells, one canperform many functional assays on the cells. The microfluidic devicesdescribed herein provide a simple, selective approach for processing ofcells.

Other features and advantages of the invention will be apparent from thefollowing description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic layout of a microfluidic device that enablesselective lysis of cells.

FIG. 2 is an illustration of the channel layout for the introduction ofthree fluids to the device, e.g., blood sample, lysis buffer, anddiluent.

FIG. 3 is an illustration of a repeating unit of the reaction chamber ofthe device where a sample of cells is passively mixed with a lysisbuffer. In one example, 133 units are connected to form the reactionchamber.

FIG. 4 is an illustration of the outlet channels of the device.

FIG. 5 is an illustration of a device for cell lysis.

FIGS. 6A and 6B are illustrations of a method for the fabrication of adevice of the invention.

FIG. 7 is a schematic diagram of a cell binding device.

FIG. 8 is an exploded view of a cell binding device.

FIG. 9 is an illustration of obstacles in a cell binding device.

FIG. 10 is an illustration of types of obstacles.

FIG. 11A is a schematic representation of a square array of obstacles.The square array has a capture efficiency of 40%. FIG. 11B is aschematic representation of an equilateral triangle array of obstacles.The equilateral triangle array has a capture efficiency of 56%.

FIG. 12A is a schematic representation of the calculation of thehydrodynamic efficiency for a square array. FIG. 12B is a schematicrepresentation of the calculation of the hydrodynamic efficiency for adiagonal array

FIGS. 13A-13B are graphs of the hydrodynamic (13A) and overallefficiency (13B) for square array and triangular array for a pressuredrop of 150 Pa/m. This pressure drop corresponds to a flow rate of 0.75mL/hr in the planar geometry.

FIG. 14A is a graph of the overall efficiency as a function of pressuredrop. FIG. 14B is a graph of the effect of the obstacle separation onthe average velocity.

FIG. 15 is a schematic representation of the arrangement of obstaclesfor higher efficiency capture for an equilateral triangular array ofobstacles in a staggered array. The capture radius r_(cap) ₂ =0.3391.The obstacles are numbered such that the first number refers to thetriangle number and the second number refers to the triangle vertex. Thestaggered array has a capture efficiency of 98%.

FIG. 16A is a graph of the percent capture of cells as a function of theflow rate for a 100 μm diameter obstacle geometry with a 50 μmedge-to-edge spacing. The operating flow regime was established acrossmultiple cell types: cancer cells, normal connective tissue cells, andmaternal and fetal samples. An optimal working flow regime is at 2.5ml/hr. FIG. 16B is a graph of the percent capture of cells as a functionof the ratio of targets cells to white blood cells. The model system wasgenerated by spiking defined number of either cancer cells, normalconnective tissue cells, or cells from cord blood into defined number ofcells from buffy coat of adult blood. The ratio of the contaminatingcells to target cells was incrementally increased 5 log with as few as10 target cells in the mixture. Yield was computed as the differencebetween number of spiked target cells captured on posts and number ofcells spiked into the sample.

FIG. 17 is an illustration of various views of the inlet and outlets ofa cell binding device.

FIG. 18 is an illustration of a method of fabricating a cell bindingdevice.

FIG. 19 is an illustration of a mixture of cells flowing through a cellbinding device.

FIG. 20A is an illustration of a cell binding device for trappingdifferent types of cells in series. FIG. 20B is an illustration of acell binding device for trapping different types of cells in parallel.

FIG. 21 is an illustration of a cell binding device that enablesrecovery of bound cells.

FIG. 22A is an optical micrograph of fetal red blood cells adhered to anobstacle of the invention. FIG. 22B is a fluorescent micrograph showingthe results of a FISH analysis of a fetal red blood cell attached to anobstacle of the invention. FIG. 22C is a close up micrograph of FIG. 22Bshowing the individual hybridization results for the fetal red bloodcell.

FIG. 23 is an illustration of a cell binding device in which beadstrapped in a hydrogel are used to capture cells.

FIG. 24A is an illustration of a device for size based separation. FIG.24B is an electron micrograph of a device for size based separation.

FIG. 25 is a schematic representation of a device of the invention forisolating and analyzing fetal red blood cells.

Figures are not necessarily to scale.

DETAILED DESCRIPTION OF THE INVENTION

The invention features methods for separating a desired cell from amixture or enriching the population of a desired cell in a mixture. Themethods are generally based on sequential processing steps, each ofwhich reduces the number of undesired cells in the mixture, but oneprocessing step may be used in the methods of the invention. Devices forcarrying out various processing steps may be separate or integrated intoone microfluidic system. The devices of the invention are a device forcell lysis, a device for cell binding, a device for arraying cells, anda device for size, shape, or deformability based separation. In oneembodiment, processing steps are used to reduce the number of cellsprior to arraying. Desirably, the methods of the invention retain atleast 75%, 80%, 90%, 95%, 98%, or 99% of the desired cells compared tothe initial mixture, while potentially enriching the population ofdesired cells by a factor of at least 100, 1000, 10,000, 100,000, oreven 1,000,000 relative to one or more non-desired cell types. Themethods of the invention may be used to separate or enrich cellscirculating in the blood (Table 1).

TABLE 1 Types, concentrations, and sizes of blood cells. Cell TypeConcentration (cells/μl) Size (μm) Red blood cells (RBC) 4.2-6.1 10 ⁶4-6  Segmented Neutrophils (WBC) 3600 >10 Band Neutrophils (WBC) 120 >10Lymphocytes (WBC) 1500 >10 Monocytes (WBC) 480 >10 Eosinophils (WBC)180 >10 Basophils (WBC) 120 >10 Platelets 500 10 ³ 1-2  Fetal NucleatedRed Blood  2-50 10 ⁻³ 8-12 Cells

Devices A. Cell Lysis

One device of the invention is employed to lysis of a population ofcells selectively, e.g., maternal red blood cells, in a mixture ofcells, e.g., maternal blood. This device allows for the processing oflarge numbers of cells under nearly identical conditions. The lysisdevice desirably removes a large number of cells prior to furtherprocessing. The debris, e.g., cell membranes and proteins, may betrapped, e.g., by filtration or precipitation, prior to any furtherprocessing.

Device.

A design for a lysis device of the invention is shown in FIG. 1. Theoverall branched architecture of the channels in the device permitsequivalent pressure drops across each of the parallel processingnetworks. The device can be functionally separated into four distinctsections: 1) distributed input channels carrying fluids, e.g., blood,lysis reagent, and wash buffer, to junctions 1 and 2 (FIG. 2); 2) aserpentine reaction chamber for the cell lysis reaction residing betweenthe two junctions (FIG. 3); 3) a dilution chamber downstream of Junction2 for dilution of the lysis reagent (FIG. 3); and 4) distributed outputchannels carrying the lysed sample to a collection vial or to anothermicrofluidic device (FIG. 4).

Input/Output Channels.

The branched input and output networks of channels enable evendistribution of the reagents into each of the channels (8, as depictedin FIG. 1). The three ports for interfacing the macro world with thedevice typically range in diameter from 1 mm-10 mm, e.g., 2, 5, 6, or 8mm. Air tight seals may be formed with ports 1, 2, and 3, e.g., throughan external manifold integrated with the device (FIG. 1). The threesolution vials, e.g., blood, lysing reagent, and diluent, may interfacewith such a manifold. The input channels from ports 1, 2, and 3 to thereaction and mixing chambers, for the three solutions shown in FIG. 1,may be separated either in the z-plane of the device (three layers, eachwith one set of distribution channels, see FIG. 2) or reside in theexternal manifold. If residing in the external manifold, thedistribution channels are, for example, CNC (computer numericallycontrolled) machined in stainless steel and may have dimensions of 500μm diameter. The manifold may hermetically interface with the device atports that are etched into locations 1′, 2′, and 3′ shown in FIG. 1.Locating the distribution channels in a manifold reduces the complexityand cost of the device. Retaining the distribution channels on thedevice will allow greater flexibility in selecting smaller channel size,while avoiding any issues of carry-over contamination between samples.Each sample input channel may have a separate output, or as depicted inFIG. 4, the output channels for each sample input are combined. As analternative to a manifold, tubing for each fluid input or output may beattached to the device, e.g., by compression fitting to gaskets ornipples or use of watertight connections such as a luer lock. Thechannels on the device transporting the fluids to the mixing junctionsand chambers beyond, can range from 10 μm-500 μm in width and depth,e.g., at most 10 μm, 25 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, 250μm, 350 μm, or 450 μm width and depth. The channel architecture isdesirably rectangular but may also be circular, semi-circular, V-shaped,or any other appropriate shape. In one embodiment, the output channel(or channels) has a cross-sectional area equal to the sum of thecross-sectional areas of the input channels.

Reaction and Dilution Chambers.

For lysis and dilution, two fluid streams are combined and allowed topass through the chambers. Chambers may be linear or serpentinechannels. In the device depicted in FIG. 1, the sample and lysis bufferare combined at junction 1, and the lysed sample and the diluent arecombined at junction 2. Serpentine architecture of the reaction chamberand dilution chamber enables sufficient resident time of the tworeacting solutions for proper mixing by diffusion or other passivemechanisms, while preserving a reasonable overall footprint for thedevice (FIG. 3). The serpentine channels may be constructed in 2D or in3D, e.g., to reduce the total length of the device or to introducechaotic advection for enhanced mixing. For short residence times, alinear chamber may be desired. Exemplary resident times include at least1 second, 5 seconds, 10 seconds, 30 seconds, 60 second, 90 seconds, 2minutes, 5 minutes, 30 minutes, 1 hour, or greater that 1 hour. The flowrate of fluids in the reaction/dilution chambers can be accuratelycontrolled by controlling the width, depth, and effective length of thechannels to enable sufficient mixing of the two reagents while enablingoptimal processing throughput. In one embodiment, the serpentine mixingchambers for cell lysis (reaction chamber) and for dilution of the lysedsample (dilution chamber) have a fluid volume each of ˜26 μl. Otherexamples of reaction/dilution chamber volumes range from 10-200 μl,e.g., at most 20, 50, 100, or 150 μl. In some embodiments, the width anddepth of the reaction and dilution chambers have the same range as theinput and output channels, i.e., 10 to 500 μm. Alternatively, thechambers may have a cross-sectional area equal to the combined areas ofany input (or output channels) in order to ensure a uniform velocity offlow through the device. In one example, the chambers are 100 μm 100 μmchannels. The total length of the chambers may be at least 1 cm, 5 cm,10 cm, 20 cm, 30 cm, 40 cm, or 50 cm.

For lysis of maternal RBCs, device output flow rates may range fromprocessing 5-16 μl of blood per second resulting in a 20-60 minuteprocessing time for 20 ml sample, or 10-30 min processing time for 10 mlsample. It is expected that the sample volume required for capturingsufficient number of fetal cells will be lower than 10 ml because of theefficiency of the process. As such, it is expected that the devicethroughput per sample will be less than 10 minutes. A residence timeof >30 seconds from the time of convergence of the two solutions,maternal blood and lysis reagent, within the passive mixer is deemedsufficient to obtain effective hemolysis (T. Maren, Mol. Pharmacol.1970, 6:430). Alternatively, the concentration of the lysis reagent canbe adjusted to compensate for residence time in the reaction chamber.The flow rates and residence times for other cell types may bedetermined by theory or experimentation. In one embodiment, the flowrates in each channel are limited to <20 μl/sec to ensure that wallshear stress on cells is less than 1 dyne/cm² (cells are known to beaffected functionally by shear stress >1 dyne/cm² though deleteriouseffects are not seen in most cells until after 10 dynes/cm²). In oneembodiment, the flow rate in each channel is at most 1, 2, 5, 10, 15μl/sec. Referring to FIG. 1, the effective length of the diluent inputchannel leading to junction 2 may be shorter than the effective lengthof the reaction chamber. This feature enables the diluent to flow intoand prime the channels downstream of junction 2, prior to arrival of thelysed sample at junction 2. The overflow buffer pre-collected in theoutput vial may act as a secondary diluent of the lysed sample whencollected, e.g., for further processing or analysis. Additionally, thediluent primes the channels downstream of junction 2 to enable smootherflow and merging of the lysed sample with the buffer in the dilutingchamber, and this priming eliminates any deleterious surface tensioneffects from dry channels on the lysed sample. The diameter of thechannels carrying the diluent may be adjusted to enable the diluent toreach junction 2 at the same time as the lysed blood to prevent anyproblems associated with air forced from the reaction chamber as thesample and lysis buffers are introduced.

Although the above description focuses on a device with eight parallelprocessing channels, any number of channels, e.g., 1, 2, 4, 16, or 32,may be employed depending on the size of the device. The device isdescribed in terms of combining two fluids for lysis and dilution, butthree or more fluids may be combined for lysis or dilution. Thecombination may be at one junction or a series of junctions, e.g., tocontrol the timing of the sequential addition of reactants. Additionalfluid inputs may be added, e.g., to functionalize the remaining cells,alter the pH, or cause undesirable components to precipitate. Inaddition, the exact geometry and dimensions of the channels may bealtered (exemplary dimensions are shown in FIG. 5). Devices of theinvention may be disposable or reusable. Disposable devices reduce therisk of contamination between samples. Reusable devices may be desirablein certain instances, and the device may be cleaned, e.g., with variousdetergents and enzymes, e.g., proteases or nucleases, to preventcontamination.

Pumping.

In one embodiment, the device employs negative pressure pumping, e.g.,using syringe pumps, peristaltic pumps, aspirators, or vacuum pumps. Thenegative pressure allows for processing of the complete volume of aclinical blood sample, without leaving unprocessed sample in thechannels. Positive pressure, e.g., from a syringe pump, peristalticpump, displacement pump, column of fluid, or other fluid pump, may alsobe used to pump samples through a device. The loss of sample due to deadvolume issues related to positive pressure pumping may be overcome bychasing the residual sample with buffer. Pumps are typically interfacedto the device via hermetic seals, e.g., using silicone gaskets.

The flow rates of fluids in parallel channels in the device may becontrolled in unison or separately. Variable and differential control ofthe flow rates in each of channels may be achieved, for example, byemploying, a multi-channel individually controllable syringe manifold.In this embodiment, the input channel distribution will be modified todecouple all of the parallel networks. The output may collect the outputfrom all channels via a single manifold connected to a suction (norequirements for an airtight seal) outputting to a collection vial or toanother microfluidic device. Alternately, the output from each networkcan be collected separately for downstream processing. Separate inputsand outputs allow for parallel processing of multiple samples from oneor more individuals.

Fabrication.

A variety of techniques can be employed to fabricate a device of theinvention, and the technique employed will be selected based in part onthe material of choice. Exemplary materials for fabricating the devicesof the invention include glass, silicon, steel, nickel,poly(methylmethacrylate) (PMMA), polycarbonate, polystyrene,polyethylene, polyolefins, silicones (e.g., poly(dimethylsiloxane)), andcombinations thereof. Other materials are known in the art. Methods forfabricating channels in these materials are known in the art. Thesemethods include, photolithography (e.g., stereolithography or x-rayphotolithography), molding, embossing, silicon micromachining, wet ordry chemical etching, milling, diamond cutting, LithographieGalvanoformung and Abformung (LIGA), and electroplating. For example,for glass, traditional silicon fabrication techniques ofphotolithography followed by wet (KOH) or dry etching (reactive ionetching with fluorine or other reactive gas) can be employed. Techniquessuch as laser micromachining can be adopted for plastic materials withhigh photon absorption efficiency. This technique is suitable for lowerthroughput fabrication because of the serial nature of the process. Formass-produced plastic devices, thermoplastic injection molding, andcompression molding is suitable. Conventional thermoplastic injectionmolding used for mass-fabrication of compact discs (which preservesfidelity of features in sub-microns) may also be employed to fabricatethe devices of the invention. For example, the device features arereplicated on a glass master by conventional photolithography. The glassmaster is electroformed to yield a tough, thermal shock resistant,thermally conductive, hard mold. This mold serves as the master templatefor injection molding or compression molding the features into a plasticdevice. Depending on the plastic material used to fabricate the devicesand the requirements on optical quality and throughput of the finishedproduct, compression molding or injection molding may be chosen as themethod of manufacture. Compression molding (also called hot embossing orrelief imprinting) has the advantages of being compatible withhigh-molecular weight polymers, which are excellent for smallstructures, but is difficult to use in replicating high aspect ratiostructures and has longer cycle times. Injection molding works well forhigh-aspect ratio structures but is most suitable for low molecularweight polymers.

A device may be fabricated in one or more pieces that are thenassembled. In one embodiment, separate layers of the device containchannels for a single fluid, as in FIG. 1. Layers of a device may bebonded together by clamps, adhesives, heat, anodic bonding, or reactionsbetween surface groups (e.g., wafer bonding). Alternatively, a devicewith channels in more than one plane may be fabricated as a singlepiece, e.g., using stereolithography or other three-dimensionalfabrication techniques.

In one embodiment, the device is made of PMMA. The features, for examplethose shown in FIG. 1, are transferred onto an electroformed mold usingstandard photolithography followed by electroplating. The mold is usedto hot emboss the features into the PMMA at a temperature near its glasstransition temperature (105° C.) under pressure (5 to 20 tons) (pressureand temperature will be adjusted to account for high-fidelityreplication of the deepest feature in the device) as shown schematicallyin FIG. 6A. The mold is then cooled to enable removal of the PMMAdevice. A second piece used to seal the device, composed of similar ordissimilar material, may be bonded onto the first piece usingvacuum-assisted thermal bonding. The vacuum prevents formation ofair-gaps in the bonding regions. FIG. 6B shows a cross-section of thetwo-piece device assembly at the junction of Port 1 (source for bloodsample) and feed channel.

Chemical Derivitization.

To reduce non-specific adsorption of cells or compounds released bylysed cells onto the channel walls, one or more channel walls may bechemically modified to be non-adherent or repulsive. The walls may becoated with a thin film coating (e.g., a monolayer) of commercialnon-stick reagents, such as those used to form hydrogels. Additionalexamples chemical species that may be used to modify the channel wallsinclude oligoethylene glycols, fluorinated polymers, organosilanes,thiols, poly-ethylene glycol, hyaluronic acid, bovine serum albumin,poly-vinyl alcohol, mucin, poly-HEMA, methacrylated PEG, and agarose.Charged polymers may also be employed to repel oppositely chargedspecies. The type of chemical species used for repulsion and the methodof attachment to the channel walls will depend on the nature of thespecies being repelled and the nature of the walls and the species beingattached. Such surface modification techniques are well known in theart. The walls may be functionalized before or after the device isassembled.

The channel walls may also be coated in order to capture materials inthe sample, e.g., membrane fragments or proteins.

Methods.

In the present invention, a sample of cells, e.g., maternal blood, isintroduced into one or more microfluidic channels. A lysis buffercontaining reagents for the selective lysis for a population of cells inthe sample is then mixed with the blood sample. Desirably, the mixingoccurs by passive means, e.g., diffusion or chaotic advection, butactive means may be employed. Additional passive and active mixers areknown in the art. The lysis reaction is allowed to continue for adesired length of time. This length of time may be controlled, forexample, by the length of the channels or by the rate of flow of thefluids. In addition, it is possible to control the volumes of solutionsmixed in the channels by altering the relative volumetric flow rates ofthe solutions, e.g., by altering the channel size or velocity of flow.The flow may be slowed down, increased, or stopped for any desiredperiod of time. After lysis has occurred, a diluent may be introducedinto the channel in order to reduce the concentration of the lysisreagents and any potentially harmful species (e.g., endosomal enzymes)released by the lysed cells. The diluent may contain species thatneutralize the lysis reagents or otherwise alter the fluid environment,e.g., pH or viscosity, or it may contain reagents for surface orintracellular labeling of cells. The diluent may also reduce the opticaldensity of the solution, which may be important for certain detectionschemes, e.g., absorbance measurements.

Exemplary cell types that may be lysed using the methods describedherein include adult red blood cells, white blood cells (such as Tcells, B cells, and helper T cells), infected white blood cells, tumorcells, and infectious organisms (e.g., bacteria, protozoa, and fungi).Lysis buffers for these cells may include cell specific IgM moleculesand proteins in the complement cascade to initiate complement mediatedlysis. Another kind of lysis buffer may include viruses that infect aspecific cell type and cause lysis as a result of replication (see,e.g., Pawlik et al. Cancer 2002, 95:1171-81). Other lysis buffers areknown in the art.

A device of the invention may be used for the selective lysis ofmaternal red blood cells (RBCs) in order to enrich a blood sample infetal cells. In this example, a maternal blood sample, 10-20 ml, isprocessed within the first one to three hours after sample collection.If the processing is delayed beyond three hours, the sample may bestored at 4° C. until it is processed. The lysis device of the inventionallows mixing of the lysis reagent (NH₄Cl (0 to 150 mM)+NaHCO₃ (0.001 to0.3 mM)+ acetazolamide (0.1 to 100 μM)) with the maternal blood toenable selective lysis of the maternal red blood cells by the underlyingprinciple of the Orskov-Jacobs-Stewart reaction (see, for example, Boyeret al. Blood 1976, 47:883-897). The high selective permeability of thecarbonic anhydrase inhibitor, acetazolamide, into fetal cells enablesselective hemolysis of the maternal red blood cells. Endogenous carbonicanhydrase in the maternal cells converts HCO₃ ⁻ to carbon dioxide, whichlyses the maternal red blood cells. The enzyme is inhibited in the fetalred blood cells, and those cells are not lysed. A diluent (e.g.,phosphate buffered saline) may be added after a period of contactbetween the lysis reagents and the cell sample to reduce the risk that aportion of the fetal red bloods cells will be lysed after prolongedexposure to the reagents.

B. Cell Binding

Another device of the invention involves depletion of whole cells from amixture by binding the cells to the surfaces of the device. The surfacesof such a device contain substances, e.g., antibodies or ligands forcell surface receptors, that bind a particular subpopulation of cells.This step in method may employ positive selection, i.e., the desiredcells are bound to the device, or it may employ negative selection,i.e., the desired cells pass through the device. In either case, thepopulation of cells containing the desired cells is collected foranalysis or further processing.

Device.

The device is a microfluidic flow system containing an array ofobstacles of various shapes that are capable of binding a population ofcells, e.g., those expressing a specific surface molecule, in a mixture.The bound cells may be directly analyzed on the device or be removedfrom the device, e.g., for further analysis or processing.Alternatively, cells not bound to the obstacles may be collected, e.g.,for further processing or analysis.

An exemplary device is a flow apparatus having a flat-plate channelthrough which cells flow; such a device is described in U.S. Pat. No.5,837,115. FIG. 7 shows an exemplary system including an infusion pumpto perfuse a mixture of cells, e.g., blood, through the microfluidicdevice. Other pumping methods, as described herein, may be employed. Thedevice may be optically transparent, or have transparent windows, forvisualization of cells during flow through the device. The devicecontains obstacles distributed, e.g., in an ordered array or randomly,throughout the flow chamber. The top and bottom surfaces of the deviceare desirably parallel to each other. This concept is depicted in FIG.8. The obstacles may be either part of the bottom or the top surface anddesirably define the height of the flow channel. It is also possible fora fraction of the obstacles to be positioned on the bottom surface, andthe remainder on the top surface. The obstacles may contact both the topand bottom of the chamber, or there may be a gap between an obstacle andone surface. The obstacles may be coated with a binding moiety, e.g., anantibody, a charged polymer, a molecule that binds to a cell surfacereceptor, an oligo- or polypeptide, a viral or bacterial protein, anucleic acid, or a carbohydrate, that binds a population of cells, e.g.,those expressing a specific surface molecule, in a mixture. Otherbinding moieties that are specific for a particular type of cell areknown in the art. In an alternative embodiment, the obstacles arefabricated from a material to which a specific type of cell binds.Examples of such materials include organic polymers (charged oruncharged) and carbohydrates. Once a binding moiety is coupled to theobstacles, a coating, as described herein, may also be applied to anyexposed surface of the obstacles to prevent non-specific adhesion ofcells to the obstacles.

A geometry of obstacles is shown in FIG. 9. In one example, obstaclesare etched on a surface area of 2 cm 7 cm on a substrate with overalldimensions of 2.5 cm 7.5 cm. A rim of 2 mm is left around the substratefor bonding to the top surface to create a closed chamber. In oneembodiment, obstacle diameter is 50 μm with a height of 100 μm.Obstacles may be arranged in a two-dimensional array of rows with a 100μm distance from center-to-center. This arrangement provides 50 μmopenings for cells to flow between the obstacles without beingmechanically squeezed or damaged. The obstacles in one row are desirablyshifted, e.g., 50 μm with respect to the adjacent rows. This alternatingpattern may be repeated throughout the design to ensure increasedcollision frequency between cells and obstacles. The diameter, width, orlength of the obstacles may be at least 5, 10, 25, 50, 75, 100, or 250μm and at most 500, 250, 100, 75, 50, 25, or 10 μm. The spacing betweenobstacles may be at least 10, 25, 50, 75, 100, 250, 500, or 750 μm andat most 1000, 750, 500, 250, 100, 75, 50, or 25 μm. Table 2 listsexemplary spacings based on the diameter of the obstacles.

TABLE 2 Exemplary spacings for obstacles. Obstacle diameter Spacingbetween (μm) obstacles (μm) 100 50 100 25 50 50 50 25 10 25 10 50 10 15

The dimensions and geometry of the obstacles may vary significantly. Forexample, the obstacles may have cylindrical or square cross sections(FIG. 10). The distance between obstacles may also vary and may bedifferent in the flow direction compared to the direction orthogonal tothe flow. In some embodiments, the distance between the edges of theobstacles is slightly larger than the size of the largest cell in themixture. This arrangement enables flow of cells without them beingmechanically squeezed between the obstacles and thus damaged during theflow process, and also maximizes the numbers of collisions between cellsand the obstacles in order to increase the probability of binding. Theflow direction with respect to the orientation of the obstacles may alsobe altered to enhance interaction of cells with obstacles.

Exemplary arrangements of obstacles are shown in FIGS. 11A-11B. Each ofthese arrangements has a calculated capture efficiency. The calculationof cell attachment considered two different geometries: a square array(FIG. 11A), and an equilateral triangular array (FIG. 11B). Overall,results are presented in terms of the efficiency of adhesion. Thecalculations consist of two parts, computing the hydrodynamic efficiency(η) and the probability of adhesion. The hydrodynamic efficiency wasdetermined as the ratio of the capture radius to the half-distancebetween the cylinders (FIGS. 12A and 12B). For the square array, η=(2r_(cap)/l)*100%, and for other arrays, η=((r_(cap1)+r_(cap2))/d₁)*100%,where d₁=d₂=l/√2 for a diagonal square array, and d₁=l√3/2, d₂=l/2 for atriangular array. The probability of adhesion represents the fraction ofcells that can resist the applied force on the cell assuming an averageof 1.5 bonds per cell and 75 pN per bond.

For the triangular array, more cells adhered to the second set ofobstacles than the first set. FIGS. 13A-13B show that the efficiencydeclines as the spacing between obstacles increases. As the spacingincreases there is a larger region outside the capture radius and thecells never contact the obstacles. Further, for the flow rates examined(0.25-1 mL/h), the overall probability of adhesion is high because theforce pr cell is less than the force to break the bonds.

For a triangular array and a spacing of 150 microns, the overallefficiency of capture drops 12% as the flow rate increases from 0.25 to1 mL/h (FIGS. 14A-14B). Adhesion is not improved by going to lower flowrates since hydrodynamic capture is not improved. The mean velocityincreases as the spacing between obstacles increases. The reason forthis is that the calculations used a constant pressure drop. Thisdiffers from the experiments in which the flow rate is held fixed andthe pressure drop varies. The results may be extrapolated from one caseto another by one skilled in the art.

A repeating triangular array provides limited capture of target cellsbecause most of the capture occurs in the first few rows. The reason forthis is that the flow field becomes established in these rows andrepeats. The first capture radius does not produce much capture whereasmost of the capture is within the second capture radius (FIG. 15). Oncecells within the capture radii are captured, the only way in whichcapture could occur is through cell-cell collisions to shift cells offtheir streamlines or secondary capture. With reference to FIG. 15, inorder to enhance capture, after the flow field is established, the rowsare shifted by a distance in the vertical direction (normal to flow) bya distance equal to r_(cap) ₂ =0.339 l. The first five columns form tworegular regions of equilateral triangles. This allows the flow to beestablished and be consistent with the solution for an equilateraltriangular array. To promote capture of cells that fall outside r_(cap)₂ , the fourth column is shifted downward by a distance r_(cap) ₂ . Allcolumns are separated by a distance equal to l/2. A cell which fallsoutside r_(cap) ₂ is shown being captured by the first obstacle in thefourth triangle (seventh column). Triangles 4 and 5 would beequilateral. In triangle 6, the vertex 3 is shifted downward by adistance r_(cap) ₂ . This arrangement may be repeated every thirdtriangle, i.e., the repeat distance is 2.5 l. FIGS. 16A and 16Billustrate the efficiency of capture as a function of flow rate andrelative population of the desired cells.

The top layer is desirably made of glass and has two slits drilledultrasonically for inlet and outlet flows. The slit inlet/outletdimensions are, for example, 2 cm long and 0.5 mm wide. FIG. 17 showsthe details for the inlet/outlet geometry. A manifold may then beincorporated onto the inlet/outlet slits. The inlet manifold acceptsblood cells from an infusion syringe pump or any other delivery vehicle,for example, through a flexible, biocompatible tubing. Similarly theoutlet manifold is connected to a reservoir to collect the solution andcells exiting the device.

The inlet and outlet configuration and geometry may be designed invarious ways. For example, circular inlets and outlets may be used. Anentrance region devoid of obstacles is then incorporated into the designto ensure that blood cells are uniformly distributed when they reach theregion where the obstacles are located. Similarly, the outlet isdesigned with an exit region devoid of obstacles to collect the exitingcells uniformly without damage.

The overall size of an exemplary device is shown in FIG. 9 (top inset).The length is 10 cm and the width is 5 cm. The area that is covered withobstacles is 9 cm 4.5 cm. The design is flexible enough to accommodatelarger or smaller sizes for different applications.

The overall size of the device may be smaller or larger, depending onthe flow throughput and the number of cells to be depleted (orcaptured). A larger device could include a greater number of obstaclesand a larger surface area for cell capture. Such a device may benecessary if the amount of sample, e.g., blood, to be processed islarge.

Fabrication.

An exemplary method for fabricating a device of the invention issummarized in FIG. 18. In this example, standard photolithography isused to create a photoresist pattern of obstacles on asilicon-on-insulator (SOI) wafer. A SOI wafer consists of a 100 μm thickSi(100) layer atop a 1 μm thick SiO₂ layer on a 500 μm thick Si(100)wafer. To optimize photoresist adhesion, the SOI wafers may be exposedto high-temperature vapors of hexamethyldisilazane prior to photoresistcoating. UV-sensitive photoresist is spin coated on the wafer, baked for30 minutes at 90° C., exposed to UV light for 300 seconds through achrome contact mask, developed for 5 minutes in developer, andpost-baked for 30 minutes at 90° C. The process parameters may bealtered depending on the nature and thickness of the photoresist. Thepattern of the contact chrome mask is transferred to the photoresist anddetermines the geometry of the obstacles.

Upon the formation of the photoresist pattern that is the same as thatof the obstacles, the etching is initiated. SiO₂ may serve as a stopperto the etching process. The etching may also be controlled to stop at agiven depth without the use of a stopper layer. The photoresist patternis transferred to the 100 μm thick Si layer in a plasma etcher.Multiplexed deep etching may be utilized to achieve uniform obstacles.For example, the substrate is exposed for 15 seconds to a fluorine-richplasma flowing SF₆, and then the system is switched to afluorocarbon-rich plasma flowing only C₄F₈ for 10 seconds, which coatsall surfaces with a protective film. In the subsequent etching cycle,the exposure to ion bombardment clears the polymer preferentially fromhorizontal surfaces and the cycle is repeated multiple times until,e.g., the SiO₂ layer is reached.

To couple a binding moiety to the surfaces of the obstacles, thesubstrate may be exposed to an oxygen plasma prior to surfacemodification to create a silicon dioxide layer, to which bindingmoieties may be attached. The substrate may then be rinsed twice indistilled, deionized water and allowed to air dry. Silane immobilizationonto exposed glass is performed by immersing samples for 30 seconds infreshly prepared, 2% v/v solution of3-[(2-aminoethyl)amino]propyltrimethoxysilane in water followed byfurther washing in distilled, deionized water. The substrate is thendried in nitrogen gas and baked. Next, the substrate is immersed in 2.5%v/v solution of glutaraldehyde in phosphate buffered saline for 1 hourat ambient temperature. The substrate is then rinsed again, and immersedin a solution of 0.5 mg/mL binding moiety, e.g., anti-CD71, anti-CD36,anti-GPA, or anti-CD45, in distilled, deionized water for 15 minutes atambient temperature to couple the binding agent to the obstacles. Thesubstrate is then rinsed twice in distilled, deionized water, and soakedovernight in 70% ethanol for sterilization.

There are multiple techniques other than the method described above bywhich binding moieties may be immobilized onto the obstacles and thesurfaces of the device. Simple physio-absorption onto the surface may bethe choice for simplicity and cost. Another approach may useself-assembled monolayers (e.g., thiols on gold) that are functionalizedwith various binding moieties. Additional methods may be used dependingon the binding moieties being bound and the material used to fabricatethe device. Surface modification methods are known in the art. Inaddition, certain cells may preferentially bind to the unaltered surfaceof a material. For example, some cells may bind preferentially topositively charged, negatively charged, or hydrophobic surfaces or tochemical groups present in certain polymers.

The next step involves the creation of a flow device by bonding a toplayer to the microfabricated silicon containing the obstacles. The topsubstrate may be glass to provide visual observation of cells during andafter capture. Thermal bonding or a UV curable epoxy may be used tocreate the flow chamber. The top and bottom may also be compression fit,for example, using a silicone gasket. Such a compression fit may bereversible. Other methods of bonding (e.g., wafer bonding) are known inthe art. The method employed may depend on the nature of the materialsused.

The cell binding device may be made out of different materials.Depending on the choice of the material different fabrication techniquesmay also be used. The device may be made out of plastic, such aspolystyrene, using a hot embossing technique. The obstacles and thenecessary other structures are embossed into the plastic to create thebottom surface. A top layer may then be bonded to the bottom layer.Injection molding is another approach that can be used to create such adevice. Soft lithography may also be utilized to create either a wholechamber made out of poly(dimethylsiloxane) (PDMS), or only the obstaclesmay be created in PDMS and then bonded to a glass substrate to createthe closed chamber. Yet another approach involves the use of epoxycasting techniques to create the obstacles through the use of UV ortemperature curable epoxy on a master that has the negative replica ofthe intended structure. Laser or other types of micromachiningapproaches may also be utilized to create the flow chamber. Othersuitable polymers that may be used in the fabrication of the device arepolycarbonate, polyethylene, and poly(methyl methacrylate). In addition,metals like steel and nickel may also be used to fabricate the device ofthe invention, e.g., by traditional metal machining. Three-dimensionalfabrication techniques (e.g., stereolithography) may be employed tofabricate a device in one piece. Other methods for fabrication are knownin the art.

Methods.

The methods of the invention involve contacting a mixture of cells withthe surfaces of a microfluidic device. A population of cells in acomplex mixture of cells such as blood then binds to the surfaces of thedevice. Desirably, at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% ofcells that are capable of binding to the surfaces of the device areremoved from the mixture. The surface coating is desirably designed tominimize nonspecific binding of cells. For example, at least 99%, 98%,95%, 90%, 80%, or 70% of cells not capable of binding to the bindingmoiety are not bound to the surfaces of the device. The selectivebinding in the device results in the separation of a specific livingcell population from a mixture of cells. Obstacles are present in thedevice to increase surface area for cells to interact with while in thechamber containing the obstacles so that the likelihood of binding isincreased. The flow conditions are such that the cells are very gentlyhandled in the device without the need to deform mechanically in orderto go in between the obstacles. Positive pressure or negative pressurepumping or flow from a column of fluid may be employed to transportcells into and out of the microfluidic devices of the invention. In analternative embodiment, cells are separated from non-cellular matter,such as non-biological matter (e.g., beads), non-viable cellular debris(e.g., membrane fragments), or molecules (e.g., proteins, nucleic acids,or cell lysates).

FIG. 19 shows cells expressing a specific surface antigen binding to abinding moiety coated onto obstacles, while other cells flow through thedevice (small arrow on cells depict the directionality of cells that arenot bound to the surface). The top and bottom surfaces of the flowapparatus may also be coated with the same binding moiety, or adifferent binding moiety, to promote cell binding.

Exemplary cell types that may be separated using the methods describedherein include adult red blood cells, fetal red blood cells,trophoblasts, fetal fibroblasts, white blood cells (such as T cells, Bcells, and helper T cells), infected white blood cells, stem cells(e.g., CD34 positive hematopoeitic stem cells), epithelial cells, tumorcells, and infectious organisms (e.g., bacteria, protozoa, and fungi).

Samples may be fractionated into multiple homogeneous components usingthe methods described herein. Multiple similar devices containingdifferent binding moieties specific for a population of cells may beconnected in series or in parallel. Serial separation may be employedwhen one seeks to isolate rare cells. On the other hand, parallelseparation may be employed when one desires to obtain differentialdistribution of various populations in blood. FIGS. 20A and 20B showparallel and serial systems for the separation of multiple populationsof cells from blood. For parallel devices, two or more sets of obstaclesthat bind different types of cells may be located in distinct regions orthey may be interspersed among each other, e.g., in a checkerboardpattern or in alternating rows. In addition, a set of obstacles may beattached to the top of the device and another set may be attached to thebottom of the device. Each set may then be derivatized to bind differentpopulations of cells. Once a sample has passed through the device, thetop and bottom may be separated to provide isolated samples of twodifferent types of cells.

The cell binding device may be used to deplete the outlet flow of acertain population of cells, or to capture a specific population ofcells expressing a certain surface molecule for further analysis. Thecells bound to obstacles may be removed from the chamber for furtheranalysis of the homogeneous population of cells (FIG. 21). This removalmay be achieved by incorporating one or more additional inlets and exitsorthogonal to the flow direction. Cells may be removed from the chamberby purging the chamber at an increased flow rate, that is higher shearforce, to overcome the binding force between the cells and theobstacles. Other approaches may involve coupling binding moieties withreversible binding properties, e.g., that are actuated by pH,temperature, or electrical field. The binding moiety, or the moleculebound on the surface of the cells, may also be cleaved by enzymatic orother chemical means.

In the example of fetal red blood cell isolation, a sample having passedthrough a lysis device is passed through a cell binding device, whosesurfaces are coated with CD45. White blood cells expressing CD45 presentin the mixture bind to the walls of the device, and the cells that passthrough the device are enriched in fetal red blood cells. Alternatively,the obstacles and device surfaces are coated with anti-CD71 in order tobind fetal nucleated red blood cells (which express the CD71 cellsurface protein) from a whole maternal blood sample. One percent ofadult white blood cells also express CD71. A sample of maternal blood ispassed through the device and both populations of cells that expressCD71 bind to the device. This results in the depletion of fetal redblood cells from the blood sample. The fetal cells are then collectedand analyzed. For example, cells are collected on a planar substrate forfluorescence in situ hybridization (FISH), followed by fixing of thecells and imaging. FIGS. 22A-22C show the use of FISH on a cell bound toan obstacle in a binding device of the invention. The cell, of fetalorigin, is stained for X and Y chromosomes using fluorescent probes.These data show the feasibility of optical imaging of FISH stained cellson posts for detection and diagnosis of chromosomal abnormalities.

Alternative Embodiments

Another embodiment of the cell binding device utilizes chemicallyderivatized glass/plastic beads entrapped in a loosely cross-linkedhydrogel, such as, but not limited to, poly(vinyl alcohol),poly(hydroxyl-ethyl methacrylate), polyacrylamide, or polyethyleneglycol (FIG. 23). The chemically derivatized beads serve as theobstacles in this embodiment. A mixture of cells is directed into thecell depletion device via two diametrically opposed inputs. Positivepressure (e.g., from an infusion pump or column of fluid) or negativepressure (e.g., from a syringe pump in pull mode, a vacuum pump, or anaspirator) drives the liquid through the hydrogel. The interaction ofthe cells in the sample with the chemically derivatized beads dispersedin the three-dimensional volume of the hydrogel results in eitherdepletion of cells, e.g., white blood cells, (negative selection) orcapture of cells, e.g., fetal red blood cells, (positive selection). Themolecular weight, cross-link density, bead density, and distribution andflow rates can be optimized to allow for maximal interaction and captureof relevant cells by the beads. The high-water content hydrogel providesa structure to trap the beads while allowing ease of flow through of thesample. The sample is then collected through two diametrically opposedoutputs. The bifurcated input/output channel design assures maximalhomogeneous distribution of the sample through the volume of thehydrogel.

In yet another embodiment, the beads are replaced by direct chemicalderivatization of the side chains of the hydrogel polymer with thebinding moiety (e.g., synthetic ligand or monoclonal antibody (mAb)).This approach can provide a very high density of molecular capture sitesand thereby assure higher capture probability. An added advantage ofthis approach is a potential use of the hydrogel based cell depletiondevice as a sensor for fetal cell capture in the positive selection mode(select for fetal cells with specific mAb), for example, if the polymerbackbone and side chain chemistry is designed to both capture the fetalcells and in the process further cross-link the hydrogel. The cells bindto numerous side chains via antigen-mAb interaction and thus serve as across-linker for the polymer chains, and the reduction in flow outputover time due to increased polymer cross-link density can bemathematically equated to the number of fetal cells captured within the3D matrix of the polymer. When the desired number of fetal cells iscaptured (measured by reduction in output flow rate), the device canstop further processing of the maternal sample and proceed to analysisof the fetal cells. The captured fetal cells can be released foranalysis by use of a photoactive coupling agent in the side chain. Thephotoreactive agent couples the target ligand or mAb to the polymerbackbone, and on exposure to a pulse of UV or IR radiation, the ligandsor mAbs and associated cells are released.

C. Cell Arraying

In this device, a mixture of cells that has typically been depleted ofunwanted cells is arrayed in a microfluidic device. An exemplary devicefor this step is described in International Publication No. WO 01/35071.The cells in the array are then assayed, e.g., by microscopy orcolorimetric assay, to locate desired cells. The desired cells may thenbe analyzed on the array, e.g., by lysis followed by PCR, or the cellsmay be collected from the array by a variety of mechanisms, e.g.,optical tweezers. In the exemplary device described in WO 01/35071, thecells are introduced into the arraying device and may passively settleinto holes machined in the device. Alternatively, positive or negativepressure may be employed to direct the cells to the holes in the array.Once the cells have been deposited in the holes, selected cells may beindividually released from the array by actuators, e.g., bubble actuatedpumps. Other methods for immobilizing and releasing cells, e.g.,dielectrophoretic trapping, may also be used in an arraying device. Oncereleased from the array, cells may be collected and subjected toanalysis. For example, a fetal red blood cell is identified in the arrayand then analyzed for genetic abnormalities. Fetal red blood cells maybe identified morphologically or by a specific molecular marker (e.g.,fetal hemoglobin, transferring receptor (CD71), thrombospondin receptor(CD36), or glycophorin A (GPA)).

D. Size-Based Separation

Another device is a device for the separation of particles based on theuse of sieves that selectively allow passage of particles based on theirsize, shape, or deformability. The size, shape, or deformability of thepores in the sieve determines the types of cells that can pass throughthe sieve. Two or more sieves can be arranged in series or parallel,e.g., to remove cells of increasing size successively.

Device.

In one embodiment, the sieve includes a series of obstacles that arespaced apart. A variety of obstacle sizes, geometries, and arrangementscan be used in devices of the invention. Different shapes of obstacles,e.g., those with circular, square, rectangular, oval, or triangularcross sections, can be used in a sieve. The gap size between theobstacles and the shape of the obstacles may be optimized to ensure fastand efficient filtration. For example, the size range of the RBCs is onthe order of 5-8 μm, and the size range of platelets is on the order of1-3 μm. The size of all WBCs is greater than 10 μm. Large gaps betweenobstacles increase the rate at which the RBCs and the platelets passthrough the sieve, but increased gap size also increases the risk oflosing WBCs. Smaller gap sizes ensure more efficient capture of WBCs butalso a slower rate of passage for the RBCs and platelets. Depending onthe type of application different geometries can be used.

In addition to obstacles, sieves may be manufactured by other methods.For example, a sieve could be formed by molding, electroforming,etching, drilling, or otherwise creating holes in a sheet of material,e.g., silicon, nickel, or PDMS. Alternatively, a polymer matrix orinorganic matrix (e.g., zeolite or ceramic) having appropriate pore sizecould be employed as a sieve in the devices described herein.

One problem associated with devices of the invention is clogging of thesieves. This problem can be reduced by appropriate sieve shapes anddesigns and also by treating the sieves with non-stick coatings such asbovine serum albumin (BSA) or polyethylene glycol (PEG), as describedherein. One method of preventing clogging is to minimize the area ofcontact between the sieve and the particles.

The schematic of a low shear stress filtration device is shown in FIG.24. The device has one inlet channel which leads into a diffuser, whichis a widened portion of the channel. Typically, the channel widens in aV-shaped pattern. The diffuser contains two sieves having pores shapedto filter, for example, smaller RBCs and platelets from blood, whileenriching the population of WBCs and fetal RBCs. The diffuser geometrywidens the laminar flow streamlines forcing more cells to come incontact with the sieves while moving through the device. The devicecontains 3 outlets, two outlets collect cells that pass through thesieves, e.g., the RBCs and platelets, and one outlet collects theenriched WBCs and fetal RBCs.

The diffuser device typically does not ensure 100% depletion of RBCs andplatelets. Initial RBC:WBC ratios of 600:1 can, however, be improved toratios around 1:1. Advantages of this device are that the flow rates arelow enough that shear stress on the cells does not affect the phenotypeor viability of the cells and that the filters ensure that all the largecells (i.e., those unable to pass through the sieves) are retained suchthat the loss of large cells is minimized or eliminated. This propertyalso ensures that the population of cells that pass through sieve do notcontain large cells, even though some smaller cells may be lost.Widening the diffuser angle will result in a larger enrichment factor.Greater enrichment can also be obtained by the serial arrangement ofmore than one diffuser where the outlet from one diffuser feeds into theinlet of a second diffuser. Widening the gaps between the obstaclesmight expedite the depletion process at the risk of losing large cellsthrough the larger pores in the sieves. For separating maternal redblood cells from fetal nucleated red blood cells, an exemplary spacingis 2-4 μm.

Method.

The device of the invention is a continuous flow cell sorter, e.g., thatfilters larger WBCs and fetal RBCs from blood. The location of thesieves in the device is chosen to ensure that the maximum number ofparticles come into contact with the sieves, while at the same timeavoiding clogging and allowing for retrieval of the particles afterseparation. In general, particles are moved across their laminar flowlines which are maintained because of extremely low Reynolds number inthe channels in the device, which are typically micrometer sized.

Fabrication.

Simple microfabrication techniques like poly(dimethylsiloxane) (PDMS)soft lithography, polymer casting (e.g., using epoxies, acrylics, orurethanes), injection molding, polymer hot embossing, lasermicromachining, thin film surface micromachining, deep etching of bothglass and silicon, electroforming, and 3-D fabrication techniques suchas stereolithography can be used for the fabrication of the channels andsieves of devices of the invention. Most of the above listed processesuse photomasks for replication of micro-features. For feature sizes ofgreater than 5 μm, transparency based emulsion masks can be used.Feature sizes between 2 and 5 μm may require glass based chromephotomasks. For smaller features, a glass based E-beam direct write maskcan be used. The masks are then used to either define a pattern ofphotoresist for etching in the case of silicon or glass or definenegative replicas, e.g., using SU-8 photoresist, which can then be usedas a master for replica molding of polymeric materials like PDMS,epoxies, and acrylics. The fabricated channels and may then be bondedonto a rigid substrate like glass to complete the device. Other methodsfor fabrication are known in the art. A device of the invention may befabricated from a single material or a combination of materials.

Example

In one example, a device for size based separation of smaller RBCs andplatelets from the larger WBCs was fabricated using simple softlithography techniques. A chrome photomask having the features andgeometry of the device was fabricated and used to pattern a siliconwafer with a negative replica of the device in SU-8 photoresist. Thismaster was then used to fabricate PDMS channel and sieve structuresusing standard replica molding techniques. The PDMS device was bonded toa glass slide after treatment with O₂ plasma. The diffuser geometry isused to widen the laminar flow streamlines to ensure that the majorityof the particles or cells flowing through the device will interact withthe sieves. The smaller RBC and platelets pass through the sieves, andthe larger WBCs are confined to the central channel.

Combination of Devices

The devices of the invention may be used alone or in any combination. Inaddition, the steps of the methods described herein may be employed inany order. A schematic representation of a combination device fordetecting and isolating fetal red blood cells is shown in FIG. 25. Inone example, a sample may be processed using the cell lysis step, andthen desired cells may be trapped in a cell binding device. If the cellstrapped are sufficiently pure, no further processing step is needed.Alternatively, only one of the lysis or binding steps may be employedprior to arraying. In another example, a mixture of cells may besubjected to lysis, size based separation, binding, and arraying.

The methods of the invention may be carried out on one integrated devicecontaining regions for cell lysis, cell binding, arraying, and sizebased separation. Alternatively, the devices may be separate, and thepopulations of cells obtained from each step may be collected andmanually transferred to devices for subsequent processing steps.

Positive or negative pressure pumping may be used to transport cellsthrough the microfluidic devices of the invention.

Analysis

After being enriched by one or more of the devices of the invention,cells may be collected and analyzed by various methods, e.g., nucleicacid analysis. The sample may also be further processed prior toanalysis. In one example, cells may be collected on a planar substratefor fluorescence in situ hybridization (FISH), followed by fixing of thecells and imaging. Such analysis may be used to detect fetalabnormalities such as Down syndrome, Edwards' syndrome, Patau'ssyndrome, Klinefelter syndrome, Turner syndrome, sickle cell anemia,Duchenne muscular dystrophy, and cystic fibrosis. The analysis may alsobe performed to determine a particular trait of a fetus, e.g., sex.

Other Embodiments

All publications, patents, and patent applications mentioned in theabove specification are hereby incorporated by reference. Variousmodifications and variations of the described method and system of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the art are intended to be within the scope of the invention.

Other embodiments are in the claims.

What is claimed is:
 1. A method for manufacturing a device comprising:i. providing a first substrate; ii. creating a pattern of obstacles onsaid first substrate, and iii. coating said pattern of obstacles withone or more binding moieties that selectively bind to one or more firstcell types in a mixture of cells, wherein said first cell type is afetal nucleated red blood cell.
 2. (canceled)
 3. The method of claim 1,wherein the substrate is selected from a silicon-based substrate, ametal-based substrate, or a polymeric-based substrate.
 4. The method ofclaim 3, wherein the silicon-based substrate is a glass substrate, asilicon substrate, or a silicon-on insulator substrate.
 5. The method ofclaim 3, wherein the metal-based substrate is a steel substrate or anickel substrate.
 6. The method of claim 3, wherein the polymeric-basedsubstrate is a charged polymer or an uncharged polymer.
 7. The method ofclaim 3, wherein the polymeric-based substrate is a urethane polymer, anepoxy polymer, an acrylic polymer, or a polymer selected from the groupconsisting of poly(methylmethacrylate), polycarbonate, polystyrene,polyethylene, poly(dimethylsiloxane) and polyolefins.
 8. The method ofclaim 1, wherein the creating step comprises the use of lithography,micromachining, casting, molding, embossing, wet chemical etch, drychemical etch, milling, diamond cutting, electroforming, LIGA, or anycombination thereof.
 9. The method of claim 8, wherein lithographycomprises photolithography, soft lithography, stereolithography or x-raylithography.
 10. The method of claim 8, wherein micromachining compriseslaser micromachining, thin-film surface micromachining, siliconmicromachining or plastic micromachining.
 11. The method of either claim8, wherein molding is injection molding.
 12. The method of either claim8, wherein the dry chemical etch is a deep reactive ion etch.
 13. Themethod of claim 1, wherein the creating step further comprises bonding asecond substrate to the first substrate.
 14. The method of claim 13,wherein the bonding comprises. the use of clamping, gluing, heating,anodic bonding, wafer bonding, a vacuum, or any combination thereof. 15.The method of claim 13, wherein the second substrate material and thefirst substrate material are the same.
 16. The method of claim 15,wherein the first substrate and the second substrate are made of apolymeric material.
 17. The method of claim 13, wherein the secondsubstrate material and the substrate material are different.
 18. Themethod of claim 13, wherein the first substrate is glass and the secondsubstrate is silicon, the first substrate is silicon and the secondsubstrate is glass, the first substrate is glass and the secondsubstrate is plastic, the first substrate is plastic and the secondsubstrate is glass, or the first substrate is plastic and the secondsubstrate a different plastic.
 19. The method of claim 1, wherein theheight of each obstacle is about 100 μm.
 20. The method of claim 1,wherein the shape of each obstacle is circular, semi-circular, oval,trapezoidal, rectangular, a square, or any combination thereof. 21-34.(canceled)